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PubMed2000

Microbial circadian oscillatory systems in Neurospora and Synechococcus: models for cellular clocks.

Common regulatory patterns have emerged among the feedback loops lying within circadian systems. Significant progress in dissecting the mechanism of clock resetting by temperature and the role of the WC proteins in the Neurospora light response has accompanied documentation of the importance of nuclear localization and phosphorylation-induced turnover of FRQ to this circadian cycle. The long-awaited molecular description of a transcription/translation loop in the Synechococcus circadian system represents a quantal step forward, followed by the identification of additional important proteins and interactions. Finally, the adaptive significance of rhythms in Synechococcus and by extension in all clocks nicely ties up an extraordinary year.

Biological Clocks↗
PubMed1970

Formate as an intermediate in the bovine rumen fermentation.

An average of 11 (range, 2 to 47) mumoles of formate per g per hr was produced and used in whole bovine rumen contents incubated in vitro, as calculated from the product of the specific turnover rate constant, k, times the concentration of intercellular formate. The latter varied between 5 and 26 (average, 12) nmoles/g. The concentration of formate in the total rumen contents was as much as 1,000 times greater, presumably owing to formate within the microbial cells. The concentration of formate in rumen contents minus most of the plant solids was varied, and from the rates of methanogenesis the Michaelis constant, K(m), for formate conversion to CH(4) was estimated at 30 nmoles/g. Also, the dissolved H(2) was measured in relation to methane production, and a K(m) of 1 nmole/g was obtained. A pure culture of Methanobacterium ruminantium showed a K(m) of 1 nmole of H(2)/g, but the K(m) for formate was much higher than the 30 nmoles for the rumen contents. It is concluded that nonmethanogenic microbes metabolize intercellular formate in the rumen. CO(2) and H(2) are the principal substrates for rumen methanogenesis. Eighteen per cent of the rumen methane is derived from formate, as calculated from the intercellular concentration of hydrogen and formate in the rumen, the Michaelis constants for conversion of these substrates by rumen liquid, and the relative capacities of whole rumen contents to ferment these substrates.

Animals↗
PubMed1992

Protein digestion and intestinal amino acids in dairy cows fed fresh Lolium perenne with different nitrogen contents.

An experiment was carried out to study digestion and intestinal AA in three ruminally and duodenally cannulated, lactating cows fed freshly cut grass (Lolium perenne) fertilized with 500 and 275 kg of N/ha per yr, respectively. High N grass was fed in June and October, and low N grass was offered in July and September. Composite samples of the grass fed in each period also were tested for in situ degradation of OM, CP, and NDF. When low N grass was fed, the digestibilities of OM and CP were lower than when high N grass was fed. On low N grass, the duodenal N flow expressed per unit of N intake was higher, although the flow of AA N on low N grass was reduced in September, mainly because of reduced microbial protein synthesis from slower OM degradation of low N grass. Duodenal N flow per unit of N intake was related negatively to the N:OM ratio of the diet. The rate of N fertilization had no effect on ruminal OM and NDF turnover rates. Turnover and passage rates in this experiment were not different from reported data on cows on winter rations with similar DMI.

Amino Acids↗
PubMed1985

Platelet-derived collagenase inhibitor: characterization and subcellular localization.

Purified human platelets were found to contain a collagenase inhibitor that is immunologically, functionally, and chromatographically identical to that produced by human skin fibroblasts. None of the other formed elements of the blood (erythrocytes, granulocytes, mononuclear cells) possessed detectable quantities of this protein. Virtually all the collagenase inhibitor contained within platelets was released following platelet activation with thrombin. Similarly, platelet activation accompanying blood clotting also resulted in the release of this protein, the ratio of plasma to serum inhibitor levels being approximately equal to 0.5. When platelets were subjected to subcellular fractionation, essentially all of the platelet-associated collagenase inhibitor was found to be located in the alpha-granule. Studies with radiolabeled inhibitor failed to detect uptake of inhibitor by platelets. Furthermore, immunologically reactive protein of similar quantity to that found in platelets was identified in human megakaryocyte lysates. Thus, the data suggest that the collagenase inhibitor is endogenously produced and stored within platelet alpha-granules. The platelet-derived collagenase inhibitor was antigenically identical to the collagenase inhibitor from human skin fibroblasts in double immunodiffusion and, like its fibroblast counterpart, inhibited collagenase on a 1:1 stoichiometric basis. When subjected to several of the chromatographic procedures utilized to purify the fibroblast protein, the platelet inhibitor behaved in an indistinguishable manner. Platelet factor 4, previously reported to be a collagenase inhibitor, was found to be immunologically unrelated to the platelet-derived collagenase inhibitor. Furthermore, platelet factor 4 displayed no collagenase inhibitory activity. Although the function of platelet-derived collagenase inhibitor is unknown, such a protein released by activated platelets may serve to regulate collagen turnover during the early stages of the inflammatory process.

Blood Platelets↗
PubMedDate not supplied

The biochemistry of collagen.

Collagen synthesis and subsequent extracellular stabilization and turnover are multi-step processes. The sequence of events in these processes is reviewed and correlated with collagen pathologies.

Animals↗
PubMed2026

Microbial succession and assembly shaped by sulfur, spatial partitioning, and water flow in a volcanic acidic river of northern Patagonia.

Extreme acidic environments represent natural laboratories for investigating the mechanisms of microbial community assembly, yet the ecological processes structuring these communities remain incompletely understood. Here, we investigate how spatial partitioning, hydrodynamics, and colonization history shape microbial succession in a unique sulfur-rich, acidic river of volcanic origin in northern Patagonia. We combined 16S rRNA gene profiling and shotgun metagenomics with a multi-scale experimental framework encompassing water column fractionation and colonization assays under native and controlled conditions. Microbial diversity was strongly influenced by spatial fractionation, with free-living communities exhibiting higher richness and temporal variability than particle-associated assemblages. Water flow modulated community structure, increasing evenness in free-living fractions under high-flow conditions, but had limited impact on particle-attached communities. Colonization of sulfur-beads followed a structured successional trajectory, with autotrophic sulfur oxidizers dominating early stages and heterotrophs adapted to biofilm lifestyles increasing over time. Ex situ recolonization assays revealed strong priority effects, with initial colonizers determining successional trajectories. Turnover analyses revealed that the balance among stochastic and deterministic assembly processes shifted across communities with pronounced stochasticity in the water column and flow-dependent effects in free-living communities, while biofilm associated communities on sulfur-beads exhibited stronger contribution of deterministic selection. These ecological patterns were mirrored by functional differentiation, with gene enrichment analyses revealing adaptive signatures of substrate attachment and resource acquisition. By integrating fine-scale environmental variation with colonization dynamics, this study reveals how microscale habitat structure and temporal fluxes jointly modulate microbial community assembly rules, offering a nuanced framework to dissect ecological processes in extreme systems.

Sulfur↗
PubMed2004

Delayed nutrient application affects mineralisation rate during composting of plant residues.

The hypothesis that delayed addition of nutrient rich material to compost would influence the mineralisation pattern was investigated by studying N turnover in compost based on wheat straw and clover-grass hay. After 712 weeks of composting almost twice as much N was mineralised when the addition of some of the N-rich clover-grass hay was postponed, suggesting that this influenced the microbial succession. The delayed addition resulted in a second temperature peak and a decline in the pH. Despite the altered conditions no significant effect was observed on the weight loss or loss of C and N. In conclusion, compost processes can in a simple way be affected by delayed substrate application leading to a higher nutrient availability without altering other parameters significantly.

Biodegradation, Environmental↗
PubMed2005

A central role for S-nitrosothiols in plant disease resistance.

Animal S-nitrosoglutathione reductase (GSNOR) governs the extent of cellular S-nitrosylation, a key redox-based posttranslational modification. Mutations in AtGSNOR1, an Arabidopsis thaliana GSNOR, modulate the extent of cellular S-nitrosothiol (SNO) formation in this model plant species. Loss of AtGSNOR1 function increased SNO levels, disabling plant defense responses conferred by distinct resistance (R) gene subclasses. Furthermore, in the absence of AtGSNOR1, both basal and nonhost disease resistance are also compromised. Conversely, increased AtGSNOR1 activity reduced SNO formation, enhancing protection against ordinarily virulent microbial pathogens. Here we demonstrate that AtGSNOR1 positively regulates the signaling network controlled by the plant immune system activator, salicylic acid. This contrasts with the function of this enzyme in mice during endotoxic shock, where GSNOR antagonizes inflammatory responses. Our data imply SNO formation and turnover regulate multiple modes of plant disease resistance.

Arabidopsis↗
PubMed1987

Monocyte procollagenase and tissue inhibitor of metalloproteinases. Identification, characterization, and regulation of secretion.

Alveolar macrophages have been shown to secrete a procollagenase and the tissue inhibitor of metalloproteinases (TIMP), which are similar or identical to the corresponding proteins of human skin fibroblasts. Little is known, however, about the collagenolytic activity of normal human monocytes. We have applied immunologic, biochemical, and molecular biologic tools to examine the collagenolytic profile of freshly isolated peripheral blood monocytes. Our studies indicate that: 1) monocytes are capable of producing both procollagenase and TIMP that are identical to the corresponding products of skin fibroblasts, alveolar macrophages, and U-937 cells; 2) unstimulated monocytes in vitro secrete high levels of TIMP, but little or no procollagenase; 3) an as yet unidentified component(s) of serum are required for in vitro production of TIMP (but not procollagenase) by monocytes; 4) even when stimulated, monocytes secrete much smaller quantities of procollagenase in comparison with macrophages; and 5) regulation of the secretion of procollagenase and TIMP by monocytes exhibits a high degree of individual variability, but is nevertheless subject to clearly different control mechanisms than our previous findings would indicate for alveolar macrophages. Monocytes thus express a macrophage-like, rather than a neutrophil-like, profile of proteins capable of mediating collagen turnover, the regulation of which is distinct from that of more differentiated alveolar macrophages. Further study of monocyte and macrophage collagenolytic activities may provide insights into both the cell biology of mononuclear phagocyte maturation and the mechanisms by which such cells mediate the turnover of interstitial collagens.

Animals↗
PubMed2004

Water pulses and biogeochemical cycles in arid and semiarid ecosystems.

The episodic nature of water availability in arid and semiarid ecosystems has significant consequences on belowground carbon and nutrient cycling. Pulsed water events directly control belowground processes through soil wet-dry cycles. Rapid soil microbial response to incident moisture availability often results in almost instantaneous C and N mineralization, followed by shifts in C/N of microbially available substrate, and an offset in the balance between nutrient immobilization and mineralization. Nitrogen inputs from biological soil crusts are also highly sensitive to pulsed rain events, and nitrogen losses, particularly gaseous losses due to denitrification and nitrate leaching, are tightly linked to pulses of water availability. The magnitude of the effect of water pulses on carbon and nutrient pools, however, depends on the distribution of resource availability and soil organisms, both of which are strongly affected by the spatial and temporal heterogeneity of vegetation cover, topographic position and soil texture. The 'inverse texture hypothesis' for net primary production in water-limited ecosystems suggests that coarse-textured soils have higher NPP than fine-textured soils in very arid zones due to reduced evaporative losses, while NPP is greater in fine-textured soils in higher rainfall ecosystems due to increased water-holding capacity. With respect to belowground processes, fine-textured soils tend to have higher water-holding capacity and labile C and N pools than coarse-textured soils, and often show a much greater flush of N mineralization. The result of the interaction of texture and pulsed rainfall events suggests a corollary hypothesis for nutrient turnover in arid and semiarid ecosystems with a linear increase of N mineralization in coarse-textured soils, but a saturating response for fine-textured soils due to the importance of soil C and N pools. Seasonal distribution of water pulses can lead to the accumulation of mineral N in the dry season, decoupling resource supply and microbial and plant demand, and resulting in increased losses via other pathways and reduction in overall soil nutrient pools. The asynchrony of resource availability, particularly nitrogen versus water due to pulsed water events, may be central to understanding the consequences for ecosystem nutrient retention and long-term effects on carbon and nutrient pools. Finally, global change effects due to changes in the nature and size of pulsed water events and increased asynchrony of water availability and growing season will likely have impacts on biogeochemical cycling in water-limited ecosystems.

Carbon↗
PubMed1986

Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains.

The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (5----10 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced overproduction of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.

Biodegradation, Environmental↗
PubMed1987

Tumor necrosis factor stimulates gelatinase and collagenase production by granulation tissue in culture.

Tumor necrosis factor (TNF, 2.6 X 10(-11)-10(-9) M) caused an increase in the production of active gelatinase and latent collagenase by granulation tissue in culture. As determined by SDS-substrate polyacrylamide gel electrophoresis, granulation tissue produced mainly two species of gelatinase with molecular weights of 64 kDa and 57 kDa, and an additional 80-kDa gelatinase was produced by TNF treatment. The results suggest that TNF may play a role in a rapid collagen turnover in the carrageenin-induced granulation tissue in rats.

Animals↗
PubMed1999

Modeling continuous bioleach reactors

The results of recent research have shown that the bioleaching of sulfide minerals occurs via a two-step mechanism. In this mechanism, the sulfide mineral is chemically oxidized by the ferric-iron in the bioleaching liquor. The ferrous-iron produced is subsequently oxidized to ferric-iron by the microorganism. Further research has shown that the rates of both the ferric leaching and ferrous-iron oxidation are governed by the ferric/ferrous-iron ratio (i.e., the redox potential). During the steady-state operation of a bioleach reactor, the rate of iron turnover between the chemical ferric leaching of the mineral and the bacterial oxidation of the ferrous-iron will define the rate and the redox potential at which the system will operate. The balance between the two rates will in turn depend on the species used, the microbial concentration, the residence time employed, the nature of the sulfide mineral being leached, and its active surface area. The model described proposes that the residence time and microbial species present determine the microbial growth rate, which in turn determines the redox potential in the bioleach liquor. The redox potential of the solution, in turn, determines the degree of leaching of the mineral; that is, conversion in the bioleach reactor. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗
PubMed1985

Content and accessibility of sialic acid on the surface of rat hepatocytes during primary culture.

The content and accessibility of terminal sialic acid and galactose residues of rat hepatocytes in primary culture were determined by in situ labeling using either periodate or sialidase/galactose oxidase treatment followed by sodium borotritiide reduction. Rat erythrocytes which were used for comparison showed a strongly enhanced tritium incorporation into galactose after sialidase treatment. In contrast, with freshly prepared rat hepatocytes only a small amount of galactose labeling was achieved after sialidase treatment. The amount of galactose labeled following sialidase treatment increased with time in culture up to day 6 and roughly paralleled the increase of the total sialic acid content. Major changes of sialic acid-containing glycoconjugates were restricted to the gangliosides. There was a transient drop in surface labeling of ganglioside-associated sialic acid on the first day in culture. The specific radioactivity of the in situ-tritiated ganglioside-sialic acid also fell by 50% in this period. Between day 2 and 4, there was an increase in gangliosidesialic acid labeling but the specific radioactivity of the sialic acid remained constant. This indicates that newly synthesized gangliosides but not the preexisting ones were accessible to periodate oxidation. The data allow conclusions about turnover and topology of the sialic acid-containing glycolipids.

Animals↗
PubMed2006

Gingival crevicular fluid matrix metalloproteinase-25 and -26 levels in periodontal disease.

BACKGROUND: Tissue destruction associated with the progression of periodontal disease is caused by a cascade of host and microbial proteolytic enzymes. Host-derived matrix metalloproteinases (MMPs) play an important role in the degradation of the extracellular matrix. Leukolysin/membrane-type 6 (MT-6)/MMP-25, the latest member of the MT-MMP subgroup of the MMP family, is primarily expressed by neutrophils and involved in extracellular matrix turnover. Matrilysin-2/MMP-26 (endometase), a novel member of the matrilysin subgroup of the MMP family, can degrade the extracellular matrix, alpha1-antitrypsin, and activate pro-MMP-9. Our study aimed to examine the levels, molecular forms, and degrees of activation of MMP-25 and MMP-26 in gingival crevicular fluid (GCF) from patients with different periodontal diseases. METHODS: A total of 105 subjects, 35 with generalized aggressive periodontitis (GAgP), 29 with chronic periodontitis (CP), 20 with gingivitis, and 21 periodontally healthy subjects, were included in this study. Periodontal status was evaluated by measuring probing depth, clinical attachment loss, presence of bleeding on probing, and plaque. GCF MMP-25 and MMP-26 levels were analyzed by computer-quantitated Western immunoblotting using specific antibodies. RESULTS: The 57-kDa soluble pro-MMP-25 and 45- to 47-kDa active form of MMP-25 were detected by Western immunoblots in CP and GAgP GCF, and lesser levels of these soluble MMP-25 immunoreactive bands were detected in gingivitis GCF. An enhanced and similar degree of MMP-25 activation was found in GAgP, CP, and gingivitis groups. There were no detectable MMP-25 immunoreactivities in the healthy subjects' GCF. GAgP and CP groups had elevated GCF MMP-26 levels and degrees of activation compared to the gingivitis and healthy groups (P <0.008). The gingivitis group had higher GCF MMP-26 levels and degree of activation compared to the healthy group (P <0.008). CONCLUSIONS: The present study demonstrated the presence of soluble or shed forms of MMP-25 and MMP-26 in GCF of patients with different periodontal diseases. Increased levels and activation of MMP-25 and MMP-26 in GCF are associated with an enhanced severity of periodontal inflammation, suggesting that these novel MMPs can participate in the progression of periodontal diseases. They may prove to be diagnostically useful and could be targets of medication in the future.

Acute Disease↗
PubMed1980

Nutrition of the weaned holstein calf. I. Effect of dietary protein level on rumen metabolism.

The effect of various dietary crude protein (CP) levels on rumen metabolism was studied with four Holstein calves (avg wt, 146 kg) fitted with ruminal and abomasal cannulae and fed four corn-based diets supplemented with 0, 5, 10 and 15% soybean meal (SBM). The diets contained 10.2, 12.2, 14.1 and 16.1% CP. Apparent rumen digestion of dry matter (DM), organic matter (OM) and starch averaged 43.9, 48.0 and 68.4%, respectively; none of these was affected by diet (P greater than .05). There was a linear increase in abomasal flow of ammonia nitrogen (N) (P less than .01) and residual N (P less than .05) as SBM increased in the diet. There was no effect of diet on abomasal flow of bacgerial N. Abomasal flow of nonammonia N (g/100 g DM intake) increased in a linear manner (P less than .05) from 1.75 at 10.2% CP to 2.07 at 16.1% CP. When protein was greater than 12% of the diet, there was evidence of wastage of protein in the rumen. Bacterial synthesis in the rumen averaged 2.0 g/100 g organic matter apparently digested. There was no apparent effect of rumen ammonia concentration on rate of microbial synthesis in spite of the low levels (less than 5 mg N/100 ml) observed with the low CP diets. There was a linear increase in serum urea levels before and after feeding as dietary CP increased (P less than .001). Volatile fatty acid concentration in the rumen, turnover rate of the fluid phase in the rumen and rumen volume were all unaffected by changes in dietary protein (P greater than .05).

Ammonia↗
PubMed1988

Selective up-regulation of human alveolar macrophage collagenase production by lipopolysaccharide and comparison to collagenase production by fibroblasts.

Collagenase catalyzes the initial and rate-limiting step in interstitial collagen degradation. Human alveolar macrophages produce both a fibroblast-like procollagenase and tissue inhibitor of metalloproteinases (TIMP). To define the potential of macrophages to express collagenase and TIMP, we have studied the effects of certain cell culture variables and LPS on in vitro production of these proteins. Our data indicate: 1) human macrophages cultured in a 1/1 (v/v) mixture of HAM F-12:DME produce two- to three-fold greater quantities of procollagenase (but not TIMP) as compared to HAM F-12, DME, or alpha-MEM alone; 2) maximal collagenase expression requires the further addition of LPS, whereas TIMP production is optimized by 5% fetal bovine serum alone; 3) the up-regulation of macrophage procollagenase by LPS represents a highly selective biologic response when compared to changes induced in other secreted and intracellular proteins; 4) measurements of steady state procollagenase mRNA by Northern blot analysis suggest that the LPS effect is mediated at a pre-translational level; and finally 5) on a per cell basis, human alveolar macrophages cultured under optional conditions secrete approximately 20% of the collagenase and approximately 10% of the TIMP elaborated by stimulated human fibroblasts. We conclude that procollagenase and TIMP secretion by human alveolar macrophages in vitro is strikingly responsive to variations in cell culture conditions and that an especially noteworthy selective upregulation of procollagenase secretion by LPS is probably modulated by a transcriptional mechanism. The macrophage synthetic potential for procollagenase suggests a potentially important role for these cells in directly mediating collagen turnover.

Adult↗
PubMed1982

Interactions in connective tissues involving monocyte/macrophages and control of production of proteinases and proteinase inhibitors.

Using human articular chondrocytes in monolayer culture as an experimental system, we have been studying mechanisms of control of production and activity of neutral proteases which degrade connective tissue matrices. Soluble factors from cultured human blood mononuclear cells (MCF) or synovial fragment cultures (SF) stimulate the production of collagenase and proteoglycanase by chondrocytes. Chondrocytes also release a collagenase inhibitor (mol. wt. 26-31,000), which is similar to the tissue inhibitor of metalloproteinases (TIMP) synthesized by cultured mammalian tissues and this is reduced in cultures exposed to MCF or SF. Retinol and dexamethasone partially inhibit the factor-stimulated collagenase, but increase the amount of inhibitor, restoring it to control levels in the presence of MCF or SF. The effects of these agents in cellular interactions in vitro will be discussed in relation to their possible roles in the control of connective tissue turnover in vivo.

Blood Proteins↗